(20S,22E)-2-methylene-19-nor-22-ene-1α,25-dihydroxyvitamin D3

ABSTRACT

This invention discloses (20S,22E)-2-methylene-19-nor-22-ene-1α,25-dihydroxyvitamin D 3  analogs, and specifically(20S,22E)-2-methylene-19-nor-22-ene-1α,25-dihydroxyvitamin D 3 , and pharmaceutical uses therefor. This compound exhibits significant vitamin D receptor binding activity and transcription activity as well as pronounced activity in arresting the proliferation of undifferentiated cells and inducing their differentiation to the monocyte thus evidencing use as an anti-cancer agent, especially for the treatment or prevention of leukemia, colon cancer, breast cancer, skin cancer or prostate cancer.

BACKGROUND OF THE INVENTION

This invention relates to vitamin D compounds, and more particularly to(20S,22E)-2-Methylene-19-nor-22-ene-1α,25-dihydroxyvitamin D₃ analogsand their pharmaceutical uses.

The natural hormone, 1α,25-dihydroxyvitamin D₁ and its analog inergosterol series, i.e. 1α,25-dihydroxyvitamin D₂ are known to be highlypatent regulators of calcium homeostasis in animals and humans, andtheir activity in cellular differentiation has also been established,Ostrem et al., Proc. Natl. Acad. Sci. USA, 84, 2610 (1987). Manystructural analogs of these metabolites have been prepared and tested,including 1α-hydroxyvitamin D₃, 1α-hydroxyvitamin D₂, various side chainhomologated vitamins and fluorinated analogs. Some of these compoundsexhibit an interesting separation of activities in cell differentiationand calcium regulation. This difference in activity may be useful in thetreatment of a variety of diseases such as renal osteodystrophy, vitaminD-resistant rickets, osteoporosis, psoriasis, and certain malignancies.

Another class of vitamin D analogs, i.e. the so called 19-nor-vitamin Dcompounds, is characterized by the replacement of the A-ring exocyclicmethylene group (carbon 19), typical of the vitamin D system, by twohydrogen atoms. Biological testing of such 19-nor-analogs (e.g.,1α,25-dihydroxy-19-nor-vitamin D₃) revealed a selective activity profilewith high potency in inducing cellular differentiation, and very lowcalcium mobilizing activity. Thus, these compounds are potentiallyuseful as therapeutic agents for the treatment of malignancies, or thetreatment of various skin disorders. Two different methods of synthesisof such 19-nor-vitamin D analogs have been described (Perlman et al.,Tetrahedron Lett, 31, 1823 (1990); Perlman et al., Tetrahedron Lett. 32,7663 (1991), and DeLuca et al., U.S. Pat. No. 5,086,191).

In U.S. Pat. No. 4,666,634, 2β-hydroxy and alkoxy (e.g., ED-71) analogsof 1α,25-dihydroxyvitamin D₃ have been described and examined by Chugaigroup as potential drugs for osteoporosis and as antitumor agents. Seealso Okano et al., Biochem. Biophys. Res. Commun. 163, 1444 (1989).Other 2-substituted (with hydroxyalkyl, e.g., ED-120, and fluoroalkylgroups) A-ring analogs of 1α,25-dihydroxyvitamin D₃ have also beenprepared and tested (Miyamoto et al., Chem. Pharm. Bull. 41, 1111(1993); Nishii et al., Osteoporosis Int, Suppl. 1, 190 (1993); Posner etal., J. Org. Chem. 59, 7855 (1994), and J. Org. Chem. 60, 4617 (1995)),as have analogs with a cyclopropyl group in the side chain (e.g. MC-903known as calcipotriene and described in Nishii et al U.S. Pat. No.5,063,221).

2-substituted analogs of 1α,25-dihydroxy-19-nor-vitamin D₃ have alsobeen synthesized, i.e. compounds substituted at 2-position with hydroxyor alkoxy groups (DeLuca et al., U.S. Pat. No. 5,536,713), with 2-alkylgroups (DeLuca et al U.S. Pat. No. 5,945,410), and with 2-alkylidenegroups (DeLuca et al U.S. Pat. No. 5,843,928), which exhibit interestingand selective activity profiles. All these studies indicate that bindingsites in vitamin D receptors can accommodate different substituents atC-2 in the synthesized vitamin D analogs.

In a continuing effort to explore the 19-nor class of pharmacologicallyimportant vitamin D compounds, analogs which are characterized by thepresence of a methylene substituent at carbon 2 (C-2), a hydroxyl groupat carbon 1 (C-1), and a shortened side chain attached to carbon 20(C-20) have also been synthesized and tested.1α-hydroxy-2-methylene-19-nor-pregnacalciferol is described in U.S. Pat.No. 6,566,352 while 1α-hydroxy-2-methylene-19-nor-homopregnacalciferolis described in U.S. Pat. No. 6,579,861 and1α-hydroxy-2-methylene-19-nor-bishomopregnacalciferol is described inU.S. Pat. No. 6,627,622. All three of these compounds have relativelyhigh binding activity to vitamin D receptors and relatively high celldifferentiation activity, but little if any calcemic activity ascompared to 1α, 25-dihydroxyvitamin D₃. Their biological activities makethese compounds excellent candidates for a variety of pharmaceuticaluses, as set forth in the '352, '861 and '622 patents.

17-ene vitamin D compounds as well as vitamin D compounds having adouble bond in the side chain thereof are also known, and have beenproposed for various pharmacological uses. Bone diseases such asosteoporosis, skin disorders such as psoriasis, cancers such as leukemiaand cosmetic conditions such as wrinkles are just some of theapplications proposed for such compounds. 17-ene compounds are describedin U.S. Pat. Nos. 5,545,633; 5,929,056 and 6,399,797 while 2-alkylidenecompounds having a side chain with a double bond therein are describedin, for example, U.S. Pat. No. 5,843,928.

19-nor vitamin D compounds substituted at the carbon-2 position of ringA with an alkyl group such as methyl, or an alkylidene group such asmethylene, and having a side chain lacking one or more of the standardvitamin D₃ substitutents, are also known, and have been proposed forvarious pharmacological uses. For example, numerous2α-methyl-19,26,27-trinor analogs are described in U.S. Pat. No.7,241,749 and in U.S. Pat. No. 7,241,909, and numerous2-methylene-19,26,27-trinor analogs are described in U.S. Pat. No.7,244,719. In addition,2α-methyl-19-nor-(20S)-1α-hydroxy-bishomopregnacalciferol is describedin published U.S. Application No. 2007/0254857, and numerous2-methylene-19,26-dinor vitamin D analogs are described in publishedU.S. Application No. 2007/0191317 and in published U.S. Application No.2007/0191316.

SUMMARY OF THE INVENTION

The present invention is directed toward(20S,22E)-2-Methylene-19-nor-22-ene-1α,25-dihydroxyvitamin D₃ analogs,their biological activity, and various pharmaceutical uses for thesecompounds. These new vitamin compounds not known heretofore are the19-nor-vitamin D analogs having a methylene group at the 2-position(C-2), a hydroxyl substituent attached to the 25-position (C-25) in theside chain, the methyl group normally located at the 21 position (C-21)in the side chain in its epi or S-configuration, and a double bondlocated between carbon atoms 22 and 23 (C-22 and C-23) in the sidechain. The preferred vitamin D analog is(20S,22E)-2-Methylene-19-nor-22-ene-1α,25-dihydroxyvitamin D₃(hereinafter referred to as “N-23”).

Structurally these(20S,22E)-2-Methylene-19-nor-22-ene-1α,25-dihydroxyvitamin D₃ analogsare characterized by the general formula I shown below:

where X₁, X₂ and X₃, which may be the same or different, are eachselected from hydrogen or a hydroxy-protecting group. The preferredanalog is (20S,22E)-2-Methylene-19-nor-22-ene-1α,25-dihydroxyvitamin D₃which has the following formula Ia:

The above compounds I, particularly Ia, exhibit a desired, and highlyadvantageous, pattern of biological activity. These compounds arecharacterized by relatively high binding to the vitamin D receptor,which is about the same as that of the native hormone1α,25-dihydroxyvitamin D₃. These compounds are more potent (one log) incausing cellular differentiation and in increasing 24-OHase geneexpression compared to 1,25(OH)₂D₃. These compounds also have lessability to promote intestinal calcium transport in vivo than1,25(OH)₂D₃, especially at the recommended lower doses. They would beclassified as having lower activity and thus lower potency in vivo instimulating intestinal calcium transport activity, as compared to thatof 1α,25-dihydroxyvitamin D₃. These compounds I, and particularly Ia,also have significant ability to mobilize calcium from bone, and theywould be classified as being slightly more effective in bone calciummobilizing activity as compared to 1α,25-dihydroxyvitamin D₃.

The above compounds I, and particularly Ia, are characterized byrelatively high cell differentiation activity and in promotingtranscription of the 24-hydroxylase gene. Thus, because these compoundsare more potent than the native hormone in causing cellulardifferentiation and transcription and are less potent in causingintestinal calcium transport, they have potential as an anti-canceragent, especially for the prevention or treatment of leukemia, coloncancer, breast cancer, skin cancer and prostate cancer.

One or more of the compounds may be present in a composition to treatthe above-noted diseases in an amount from about 0.01 μg/gm to about1000 μg/gm of the composition, preferably from about 0.1 μg/gm to about500 μg/gm of the composition, and may be administered topically,transdermally, orally, rectally, nasally, sublingually or parenterallyin dosages of from about 0.01 μg/day to about 1000 μg/day, preferablyfrom about 0.1 μg/day to about 500 μg/day.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings:

FIGS. 1-5 illustrate various biological activities of(20S,22E)-2-Methylene-19-nor-22-ene-1α,25-dihydroxyvitamin D₃,hereinafter referred to as “N-23”, as compared to the native hormone1α,25-dihydroxyvitamin D₃, hereinafter “1,25(OH)₂D₃.”

FIG. 1 is a graph illustrating the relative activity of N-23 and1,25(OH)₂D₃ to compete for binding with [³H]-1,25-(OH)₂-D₃ to thefull-length recombinant rat vitamin D receptor;

FIG. 2 is a graph illustrating the percent HL-60 cell differentiation asa function of the concentration of N-23 and 1,25(OH)₂D₃;

FIG. 3 is a graph illustrating the in vitro transcription activity of1,25(OH)₂D₃ as compared to N-23;

FIG. 4 is a graph illustrating the bone calcium mobilization activity of1,25(OH)₂D₃ as compared to N-23 in a group of animals; and

FIG. 5 is a graph illustrating the intestinal calcium transport activityof 1,25(OH)₂D₃ as compared to N-23 in a group of animals.

DETAILED DESCRIPTION OF THE INVENTION

(20S,22E)-2-Methylene-9-nor-22-ene-1α,25-dihydroxyvitamin D₃ (referredto herein as “N-23”) a 19-nor vitamin D analog which is characterized bythe presence of a methylene substituent at the carbon 2 (C-2), ahydroxyl substituent attached to the 25-position (C-25) in the sidechain, the methyl group normally located at the 21 position (C-21) inthe side chain in its epi or S-configuration, and a double bond locatedbetween carbon atoms 22 and 23 (C-22 and C-23) in the side chain, wassynthesized and tested. Such vitamin D analog seemed an interestingtarget because the relatively small methylene group at the C-2 positionshould not interfere with binding to the vitamin D receptor,Structurally, this 19-nor analog is characterized by the general formulaIa previously illustrated herein, and its pro-drug (in protected hydroxyform) is characterized by general formula I previously illustratedherein.

The preparation of(20S,22E)-2-Methylene-19-nor-22-ene-1α,25-dihydroxyvitamin D₃ analogshaving the structure I can be accomplished by a common general method,i.e. the condensation of a bicyclic Windaus-Grundmann type ketone IIwith the allylic phosphine oxide III to the corresponding2-methylene-19-nor-vitamin D analog IV followed by deprotection at C-1,C-3 and C-25 in the latter compound (see Schemes 1 and 2 herein):

In the structures II, III and IV, groups X₁, X₂ and X₃ arehydroxy-protecting groups, preferably t-butyldimethylsilyl, it beingalso understood that any functionalities that might be sensitive, orthat interfere with the condensation reaction, be suitably protected asis well-known in the art. The process shown above represents anapplication of the convergent synthesis concept, which has been appliedeffectively for the preparation of vitamin D compounds [e.g. Lythgoe etal., J. Chem. Soc. Perkin Trans. I, 590 (1978); Lythgoe, Chem. Soc. Rev.9, 449 (1983); Toh et al., J. Org. Chem. 48, 1414 (1983); Baggiolini etal., J. Org. Chem. 51, 3098 (1986); Sardina et al., J. Org. Chem. 51,1264 (1986); J. Org. Chem. 51, 1269 (1986); DeLuca et al., U.S. Pat. No.5,086,191; DeLuca et al., U.S. Pat. No. 5,536,713].

The hydrindanone of the general structure II is not known. It can beprepared by the method shown in Schemes 1 and 2 herein (see thepreparation of compound N-23).

For the preparation of the required phosphine oxides of generalstructure III, a synthetic route has been developed starting from amethyl quinicate derivative which is easily obtained from commercial(1R,3R,4S,5R)-(−)-quinic acid as described by Perlman et al.,Tetrahedron Lett. 32, 7663 (1991) and DeLuca et al., U.S. Pat. No.5,086,191.

The overall process of the synthesis of compounds I and Ia isillustrated and described more completely in U.S. Pat. No. 5,843,928entitled “2-Alkylidene-19-Nor-Vitamin D Compounds” the specification ofwhich is specifically incorporated herein by reference.

As used in the description and in the claims, the term“hydroxy-protecting group” signifies any group commonly used for thetemporary protection of hydroxy functions, such as for example,alkoxycarbonyl, acyl, alkylsilyl or alkylarylsilyl groups (hereinafterreferred to simply as “silyl” groups), and alkoxyalkyl groups.Alkoxycarbonyl protecting groups are alkyl-O—CO— groupings such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl,butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl,benzyloxycarbonyl or allyloxycarbonyl. The term “acyl” signifies analkanoyl group of 1 to 6 carbons, in all of its isomeric forms, or acarboxyalkanoyl group of 1 to 6 carbons, such as an oxalyl, malonyl,succinyl, glutaryl group, or an aromatic acyl group such as benzoyl, ora halo, nitro or alkyl substituted benzoyl group. The word “alkyl” asused in the description or the claims, denotes a straight-chain orbranched alkyl radical of 1 to 10 carbons, in all its isomeric forms.“Alkoxy” refers to any alkyl radical which is attached by oxygen, i.e. agroup represented by “alkyl-O.”Alkoxyalkyl protecting groups aregroupings such as methoxymethyl, ethoxymethyl, methoxyethoxymethyl, ortetrahydrofuranyl and tetrahydropyranyl. Preferred silyl-protectinggroups are trimethylsilyl, triethylsilyl, t-butyldimethylsilyl,dibutylmethylsilyl, diphenylmethylsilyl, phenyldimethylsilyl,diphenyl-t-butylsilyl and analogous alkylated silyl radicals. The term“aryl” specifies a phenyl-, or an alkyl-, nitro- or halo-substitutedphenyl group.

A “protected hydroxy” group is a hydroxy group derivatised or protectedby any of the above groups commonly used for the temporary or permanentprotection of hydroxy functions, e.g. the silyl, alkoxyalkyl, acyl oralkoxycarbonyl groups, as previously defined. The terms “hydroxyalkyl”,“deuteroalkyl” and “fluoroalkyl” refer to an alkyl radical substitutedby one or more hydroxy, deuterium or fluoro groups respectively. An“alkylidene” refers to a radical having the general formula C_(k)H_(2k)—where k is an integer.

More specifically, reference should be made to the followingillustrative example and description as well as to Schemes 1 and 2herein for a detailed illustration of the preparation of compound N-23.

In this example specific products identified by Arabic numerals (1, 2,3) refer to the specific structures so identified in the Schemes 1 and2.

EXAMPLES

Chemistry. Ultraviolet (UV) absorption spectra were recorded with aHitachi Model 60-100 UV-vis spectrometer in the solvent noted. ¹Hnuclear magnetic resonance (NMR) spectra were recorded at 500 MHz with aBruker AM-500 FT spectrometer in deuterochloroform. Chemical shifts (δ)are reported downfield from internal Me₄Si (δ 0.00). Mass spectra wererecorded at 70 eV on a Kratos DS-50 TC instrument equipped with a KratosMS-55 data system. Samples were introduced into the ion sourcemaintained at 120-250° C. via a direct insertion probe. High-performanceliquid chromatography (HPLC) was performed on a Waters Associates liquidchromatograph equipped with a Model 6000A solvent delivery system, aModel 6 UK Universal injector, a Model 486 tunable absorbance detector,and a differential R 401 refractometer.

Example 1 Preparation of(8S,20S)-des-A,B-20-(hydroxymethyl)-pregnan-8-ol (1)

Ozone was passed through a solution of vitamin D₂ (3 g, 7.6 mmol) inmethanol (250 mL) and pyridine (2.44 g, 2.5 mL, 31 mmol) for 50 min at−78° C. The reaction mixture was then flushed with an oxygen for 15 minto remove the residual ozone and the solution was treated with NaBH₄(0.75 g, 20 mmol). After 20 min the second portion of NaBH₄ (0.75 g, 20mmol) was added and the mixture was allowed to warm to room temperature.The third portion of NaBH₄ (0.75 g, 20 mmol) was then added and thereaction mixture was stirred for 18 h. The reaction was quenched withwater (40 mL) and the solution was concentrated under reduced pressure.The residue was extracted with ethyl acetate and the combined organicphases were washed with 1 M aq. HCl, saturated aq. NaHCO₃, dried(Na₂SO₄) and concentrated under reduced pressure. The residue waschromatographed on silica gel with hexane/ethyl acetate (75:25) to givethe diol 1 (1.21 g, 75% yield) as white crystals:

m.p. 106-108° C.; [α]_(D)+30.2° (c 1.46, CHCl₃); ¹H NMR (400 MHz, CDCl₃)δ 4.08 (1H, d, J=2.0 Hz, 8α-H), 3.63 (1H, dd, J=10.5, 3.1 Hz, 22-H),3.38 (1H, dd, J=10.5, 6.8 Hz, 22-H), 1.99 (1H, br.d, J=13.2 Hz), 1.03(3H, d, J=6.6 Hz, 21-H₃), 0.956 (3H, s, 18-H₃); ¹³C NMR (100 MHz) δ69.16 (d, C-8), 67.74 (t, C-22), 52.90 (d), 52.33 (d), 41.83 (s, C-13),40.19 (t), 38.20 (d), 33.53 (t), 26.62 (t), 22.54 (t), 17.36 (t), 16.59(q, C-21), 13.54 (q, C-18); MS (EI) m/z 212 (2, M^(+),) 194 (34,M⁺-H₂O), 179 (33, M⁺-H₂O—CH₃), 163 (18, M⁺CH₂OH—H₂O), 135 (36), 125(54), 111 (100), 95 (63), 81 (67); exact mass calculated for C₁₃H₂₂O(M⁺-H₂O) 194.1671, found 194.1665.

Preparation of (8S,20S)-des-A,B-8-benzoyloxy-20-(hydroxymethyl)-pregnane(2)

Benzoyl chloride (2.4 g, 2 mL, 17 mmol) was added to a solution of thediol 1 (1.2 g, 5.7 mmol) and DMAP (30 mg, 0.2 mmol) in anhydrouspyridine (20 mL) at 0° C. The reaction mixture was stirred at 4° C. for24 h, diluted with methylene chloride (100 mL), washed with 5% aq. HCl,water, saturated aq. NaHCO₃, dried (Na₂SO₄) and concentrated underreduced pressure. The residue (3.39 g) was treated with a solution ofKOH (1 g, 15.5 mmol) in anhydrous ethanol (30 mL) at room temperature.After stirring of the reaction mixture for 3 h, ice and 5% aq. HCl wereadded until pH=6. The solution was extracted with ethyl acetate (3×50mL) and the combined organic phases were washed with saturated aq.NaHCO₃, dried (Na₂SO₄) and concentrated under reduced pressure. Theresidue was chromatographed on silica gel with hexane/ethyl acetate(75:25) to give the alcohol 2 (1.67 g, 93% yield) as a colorless oil:

[α]_(D)+56.0 (c 0.48, CHCl₃); ¹H NMR (400 MHz, CDCl₃ αTMS) δ 8.08-8.02(2H, m, o-H_(Bz)), 7.59-7.53 (1H, m, p-H_(Bz)), 7.50-7.40 (2H, m,m-H_(Bz)), 5.42 (1H, d, 2.4 Hz, 8α-H), 3.65 (1H, dd, J=10.5, 3.2 Hz,22-H), 3.39 (1H, dd, J=10.5, 6.8 Hz, 22-H), 1.08 (3H, d, J=5.3 Hz,21-H₃), 1.07 (3H, s, 18-H₃); ¹³C NMR (125 MHz) δ 166.70 (s, C═O), 132.93(d, p-C_(Bz)), 130.04 (s, i-C_(Bz)), 129.75 (d, o-C_(Bz)), 128.57 (d,m-C_(Bz)), 72.27 (d, C-8), 67.95 (t, C-22), 52.96 (d), 51.60 (d), 42.15(s, C-13), 39.98 (t), 38.61 (d), 30.73 (t), 26.81 (t), 22.91 (t), 18.20(t), 16.87 (q, C-21), 13.81 (q, C-18); MS (EI) m/z 316 (5, M⁺), 301 (3,M⁺-Me), 299 (1, M⁺-OH), 298 (2, M⁺-H₂O), 285 (10, M⁺-CH₂OH), 257 (6),230 (9), 194 (80), 135 (84), 105 (100); exact mass calculated forC₂₀H₂₈O₃ 316.2038, found 316.2019.

Preparation of (8S,20S)-des-A,B-8-benzoyloxy-20-formyl-pregnane (3)

Sulfur trioxide pyridine complex (1.94 g, 12.2 mmol) was added to asolution of the alcohol 2 (640 mg, 2.03 mmol), triethylamine (1.41 mL,1.02 g, 10.1 mmol) in anhydrous methylene chloride (10 mL) and anhydrousDMSO (2 mL) at 0° C. The reaction mixture was stirred under argon at 0°C. for 1 h and then concentrated. The residue was diluted with ethylacetate, washed with brine, dried (Na₂SO₄) and concentrated. The residuewas purified by column chromatography on silica gel with hexane/ethylacetate (95:5) to give the aldehyde 3 (529 mg, 83% yield) as an oil:

[α]_(D)+63.1 (c 5.85, CHCl₃); ¹H NMR (400 MHz, CDCl₃+TMS) δ 9.60 (1H, d,J=3.1 Hz, CHO, 8.05 (2H, m, o-H_(Bz),) 7.57 (1H, m, p-H_(Bz)), 7.45 (2H,m, m-H_(Bz)), 5.44 (1H, s, 8α-H), 2.39 (1H, m, 20-H), 2.03 (2H, dm,J=11.5 Hz), 1.15 (3H, d, J=6.9 Hz, 21-H₃), 1.10 (3H, s, 18-H₃); ¹³C NMR(100 MHz) δ 204.78 (d, CHO), 166.70 (s, C═O), 132.78 (d, p-Bz), 130.69(s, i-Bz), 129.50 (d, o-Bz), 128.38, (d, m-Bz), 71.66 (d, C-8), 51.30(d), 50.95 (d), 49.20 (d), 42.38 (s, C-13), 39.62 (t), 30.47 (t), 25.99(t), 22.92 (t), 17.92 (t), 13.90 (q), 13.35 (q); MS (EI) m/z 314 (1,M⁺), 299 (0.5, M⁺-Me), 286 (1, M⁺-CO), 285 (5, M⁺-CHO), 257 (1,M⁺C₃H_(S)O), 209 (10, M⁺-PhCO), 192 (38), 134 (60), 105 (100), 77 (50);exact mass calculated for C₂₀H₂₆O₃314.1882, found 314.1887.

Preparation of (8S,20R)-des-A,B-8-benzoyloxy-20-(hydroxymethyl)-pregnane(4)

The aldehyde 3 (364 mg, 1.12 mmol) was dissolved in methylene chloride(15 mL) and a 40% aq. n-Bu₄NOH solution (1.47 mL, 1.45 g, 2.24 mmol) wasadded. The resulting mixture was stirred under argon at room temperaturefor 16 h, diluted with methylene chloride (20 mL), washed with water,dried (Na₂SO₄) and concentrated under reduced pressure. A residue waschromatographed on silica gel with hexane/ethyl acetate (95:5) to afforda mixture of aldehyde 3 and its 20-epimer (292 mg, 80% yield) in ca. 1:2ratio (by ¹H NMR).

This mixture of aldehydes (292 mg, 0.9 mmol) was dissolved in THF (5 mL)and NaBH₄ (64 mg, 1.7 mmol) was added, followed by a dropwise additionof ethanol (5 mL). The reaction mixture was stirred at room temperaturefor 30 min and it was quenched with a saturated aq. NH₄Cl solution. Themixture was extracted with ether (3×20 mL) and the combined organicphase was washed with water, dried (Na₂SO₄) and concentrated underreduced pressure. The residue was chromatographed on silica gel withhexane/ethyl acetate (96:4→80:20) to give the desired, pure(20R)-alcohol 4 (160 mg, 55% yield) as an oil and a mixture of 4 and its20-epimer 2 (126 mg, 43% yield) in ca. 1:3 ratio (by ¹H NMR).

[α]_(D)+50.1 (c 1.09, CHCl₃); ¹H NMR (400 MHz, CDCl₃+TMS) δ 8.05 (2H, m,o-H_(Bz)), 7.55 (1H, m, p-H_(Bz)), 7.44 (2H, m, m-H_(Bz)), 5.41 (1H, s,8α-H), 3.77 (1H, dd, J=10.4, 3.3 Hz, 22-H), 3.45 (1H, dd, J=10.4, 7.4Hz, 22-H), 1.067 (3H, s, 18-H₃), 0.973 (3H, d, J=6.6 Hz, 21-H₃); ¹³C NMR(100 MHz) δ 166.36 (s, C═O), 132.61 (d, p-C_(Bz)), 130.63 (s, i-C_(Bz)),129.39 (d, o-C_(Bz)), 128.23 (d, m-C_(Bz)), 71.97 (d, C-8), 66.42 (t,C-22), 52.65 (d), 51.38 (d), 41.58 (s, C-13), 39.16 (t), 37.45 (d),30.38 (t), 26.29 (t),22.35 (t), 17.89 (t), 16.42 (q, C-21), 13.78 (q,C-18); MS (EI) m/z 316 (16, M⁺), 301 (5, M⁺-Me), 299 (2, M⁺-OH), 298 (3,M⁺-H₂O), 285 (9, M⁺-CH₂OH), 257 (5), 242 (11), 230 (8), 194 (60), 147(71), 105 (100); exact mass calculated for C₂₀H₂₈O₃ 316.2038, found316.2050.

Preparation of (8S,20R)-des-A,B-8-benzoyloxy-20-formyl-pregnane (5)

Sulfur trioxide pyridine complex (258 mg, 1.62 mmol) was added to asolution of the alcohol 4 (111 mg, 0.35 mmol), triethylamine (188)μL,136 mg, 1.35 mmol) in anhydrous methylene chloride (5 mL) and anhydrousDMSO (1 mL) at 0° C. The reaction mixture was stirred under argon at 0°C. for 1 h and then concentrated. The residue was diluted with ethylacetate, washed with brine, dried (Na₂SO₄) and concentrated. The residuewas purified by column chromatography on silica gel with hexane/ethylacetate (95:5) to give the aldehyde 5 (93 mg, 85% yield) as an oil:

[α]_(D)+28.8 (c 0.88, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 9.55 (1H, d,J=5.0 Hz, CHO), 8.02 (2H, m, o-H_(Bz)), 7.54 (1H, m, p-H_(Bz)), 7.43(2H, m, m-H_(Bz)), 5.42 (1H, s, 8α-H), 2.35 (1H, m, 20-H), 2.07 (1H, m),1.87 (1H, m), 1.05 (3H, s, 18-H₃), 1.04 (3H, d, J=7.8 Hz, 21-H₃); ¹³CNMR (125 MHz) δ 205.51 (d, CHO), 166.34 (s, C═O), 132.76 (d, p-C_(Bz)),130.62 (s, i-C_(Bz)), 129.47 (d, o-C_(Bz)), 128.35, (d, m-C_(Bz)), 71.52(d, C-8), 52.08 (d), 51.08 (d), 48.40 (d), 41.55 (s, C-13), 38.54 (t),30.41 (t), 25.28 (t), 22.08 (t), 17.68 (t), 14.49 (q), 13.38 (q); MS(EI) m/z 314 (2, M⁺), 285 (3, M⁺-CHO), 209 (8, M⁺PhCO), 192 (30,M⁺PhCOOH), 177 (14), 134 (45), 105 (100), 77 (50); exact mass calculatedfor C₁₉H₂₅O₂(M⁺-CHO) 285.1855, found 285.1849.

Preparation of(8S,20S)-des-A,B-8-benzoyloxy-20-[4′-hydroxy-4′-methyl-pent-(1′E)-en-yl]-pregnane(7)

n-Butyllithium (1.61 M, 1.38 mL, 2.22 mmol) was added to a stirredsuspension of the phosphonium salt 6 (476 mg, 1.11 mmol) in anhydrousTHF (6 mL) at −20° C. The solution turned orange. After 1 h a precooled(−20° C.) solution of the aldehyde 5 (93 mg, 0.30 mmol) in anhydrous THF(1+0.5 mL) was added and the reaction mixture was stirred at −20° C. for3 h and at room temperature for 18 h. The reaction was quenched withwater and the mixture was extracted with ethyl acetate. Combined organicphases were washed with brine, dried (Na₂SO₄) and evaporated. Theresidue was chromatographed on silica gel with hexane/ethyl acetate(95:5, then 90:10) to give the product 7 (52 mg, 45% yield):

[α]_(D)-25.1 (c 2.5, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 8.04 (2H, m,o-H_(Bz)), 7.55 (1H, m, p-H_(Bz)), 7.44 (2H, m, m-H_(Bz)), 5.42 (3H, m,8α-H, 22-H, 23-H), 1.22 (6H, 26,27-H₆), 1.04 (3H, s, 18-H₃), 0.94 (3H,d, J=6.6 Hz, 21-H₃); ¹³C NMR (125 MHz) δ 166.41 (s, C═O), 141.34 (d,C-22), 132.64 (d, p-C_(Bz)), 130.83 (s, i-C_(Bz)), 129.50 (d, o-C_(Bz)),128.29 (d, m-C_(Bz)), 122.86 (d, C-23), 72.06 (d, C-8), 70.68 (s, C-25),56.30 (d), 51.46 (d), 46.92 (t), 41.91 (s, C-13), 40.23 (d), 39.33 (t),30.57 (t), 29.12 and 29.11 (each q, C-26 and C-27), 26.83 (t), 22.49(t), 21.57 (q, C-21), 17.78 (t), 13.80 (q, C-18); MS (EI) m/z no M⁺, 366(5, M⁺-H₂O), 326 (38, M⁺-C₃H₆O), 284 (38, M⁺-C₆H₁₂O), 204 (54), 162(88), 135 (93), 121 (53), 105 (100); exact mass calculated forC₂₅H₃₆O₃Na (MNa⁺) 407.2562, found 407.2561.

Preparation of(8S,20S)-des-A,B-8-benzoyloxy-20-[4′-(tert-butyldimethylsilyloxy)-4′-methyl-pent-(1′E)-en-yl]-pregnane(8)

tert-Butyldimethylsilyi trifluoromethanesulfonate (32 μL, 37 mg, 0.14mmol) was added to a solution of the alcohol 7 (52 mg, 0.14 mmol) and2,6-lutidine (33 μL, 30 mg, 0.28 mmol) in anhydrous methylene chloride(3 mL) at −20° C. The mixture was stirred under argon at roomtemperature for 3 h. The reaction was quenched with water and extractedwith methylene chloride. The combined organic phases were washed withbrine, dried (Na₂SO₄) and concentrated under reduced pressure. Theresidue was chromatographed on silica gel with hexane and hexane/ethylacetate (97:3) to give the product 8 (65 mg, 93%):

[α]D−21.2 (c 4.95, CHCl₃); ¹H NMR (500 MHz, CDCl₃+TMS) δ 8.05 (2H, m,o-H_(Bz)), 7.54 (1H, m, p-H_(Bz)), 7.43 (2H, m, m-H_(Bz)), 5.41 (2H, m,8α-H and 23-H), 5.29 (1H, dd, J=15.4, 9.1 Hz, 22-H), 1.18 (6H, d, J=4.5Hz, 26,27-H₆), 1.04 (3H, s, 18-H₃), 0.93 (3H, d, J=6.6 Hz, 21-H₃), 0.87(9H, s, Si-t-Bu), 0.08 (6H, s, SiMe₂); ¹³C NMR (125 MHz) δ 166.43 (s,C═O), 139.10 (d, C-22), 132.62 (d, p-C_(Bz)), 130.91 (s, i-C_(Bz)),129.53 (d, o-C_(Bz)), 128.30 (d, m-C_(Bz)), 124.39 (d, C-23), 73.72 (s,C-25), 72.14 (d, C-8), 56.44 (d), 51.52 (d), 48.29 (t), 41.94 (s, C-13),40.30 (d), 39.28 (t), 30.63 (t), 29.76 and 29.65 (each q, C-26 andC-27), 26.88 (t), 25.83 (q, SiCMe₃ ), 22.56 (t), 21.53 (q, C-21), 18.04(s, SiCMe₃), 17.82 (t), 13.68 (q, C-18), −2.04 (q, SiMe₂ ); MS (EI) m/zno M⁺, 483 (5, M⁺-CH₃), 441 (8, M⁺-C₄H₉), 359 (2), 339 (5), 245 (6), 237(9), 173 (100), 163 (36), 135 (38), 105 (54); exact mass calculated forC₃₁H₅₀O₃SiNa (MNa⁺) 521.3427, found 521.3450.

Preparation of(8S,20S)-des-A,B-20-[4′-(tert-butyldimethylsilyloxy)-4′-methyl-pent-(1′E)-en-yl]-pregnan-8-ol(9)

A solution of sodium hydroxide in ethanol (2.5M, 8 mL) was added to astirred solution of the benzoate 8 (65 mg, 131 μmol) in anhydrousethanol (20 mL) and the reaction mixture was refluxed for 21 h. Themixture was cooled to room temperature, neutralized with 5% aq. HCl andextracted with dichloromethane. Combined organic phases were washed withsaturated aq. NaHCO₃, dried (Na₂SO₄) and evaporated. The residue waschromatographed on silica gel with hexane/ethyl acetate (97:3) to givethe alcohol 9 (49 mg, 95% yield):

[α]_(D)+1.1 (c 3.6, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 5.38 (1H, ddd,J=15.4, 7.9, 7.0 Hz, 23-H), 5.25 (1H, dd, J=15.4, 9.1 Hz, 22-H), 4.07(1H, s, 8α-H), 2.10 (3H, m), 1.16 (6H, d, J=3.0 Hz, 26,27-H₆), 0.92 (3H,s, 18-H₃), 0.89 (3H, d, J=6.7 Hz, 2-H₃), 0.86 (9H, s, Si-t-Bu), 0.07(6H, s, SiMe₂); ¹³C NMR (125 MHz) δ 139.15 (d, (C-22), 124.27 (d, C-23),73.73 (s, C-25), 69.35 (d, C-8), 56.69 (d), 52.53 (d), 48.28 (t), 41.86(s, C-13), 40.06 (d), 39.71 (t), 33.66 (t), 29.77 and 29.61 (each q,C-26 and C-27), 26.93 (t), 25.82 (q, SiCMe₃ ), 22.39 (t), 21.51 (q,C-21), 18.04 (s, SiCMe₃), 17.22 (t), 13.68 (q, C-18), −2.05 (q, SiMe₂ );MS (EI) m/z M⁺, 393 (0.5, M⁺-H), 379 (5, M⁺-CH₃), 337 (3, M⁺-C₄H₉), 279(6, M⁺-t-BuSiMe₂), 255 (9), 237 (96), 193 (42), 173 (100), 157 (43), 135(40), 115 (37); exact mass calculated for C₂₄H₄₆O₂SiNa (MNa⁺) 417.3165,found 417.3165.

Preparation of(20S)-des-A,B-20-[4′-tert-butyldimethylsilyloxy)-4′-methyl-pent-(1′E)-en-yl]-pregnan-8-one(10)

Molecular sieves A4 (0.6 g) were added to a solution of4-methylmorpholine oxide (55 mg, 0.47 mmol) in dichloromethane (0.6 mL).The mixture was stirred at room temperature for 15 min andtetrapropylammonium perruthenate (8 mg, 23 μmol) was added, followed bya solution of alcohol 9 (28 mg, 71 μmol) in dichloromethane (300+250μL). The resulting suspension was stirred at room temperature for 1 h.The reaction mixture was filtered through a Waters silica Sep-Pakcartridge (5 g) that was further washed with dichloromethane. Afterremoval of the solvent the ketone 10 (27 mg, 97% yield) was obtained asa colorless oil:

[α]_(D)-39.3 (c 1.35, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 5.42 (1H, ddd,J=15.4, 8.2, 7.0 Hz, 23-H), 5.28 (1H, dd, J=15.4, 9.1 Hz, 22-H), 2.42(1H, dd, J=11.4, 7.6 Hz), 1.17 (6H, s, 26,27-H₆), 0.94 (3H, d, J=6.6 Hz,21-H₃), 0.86 (9H, s, Si-t-Bu), 0.61 (3H, s, 18-H₃), 0.069 and 0.065(each 3H, each s, each SiMe₂); ¹³C NMR (100 MHz) δ 212.10 (s, C═O),138.69 (d, C-22), 124.97 (d, C-23), 73.65 (s, C-25), 61.91 (d), 56.57(d), 50.03 (s, C-13), 48.23 (t), 41.03 (t), 40.44 (d), 38.28 (t), 29.79and 29.62 (each q, C-26 and C-27), 27.21 (t), 25.79 (q, SiCMe₃ ), 23.93(t), 21.52 (q, C-21), 18.97 (t), 18.03 (s, SiCMe₃), 12.49 (q, C-18),−2.06 (q, SiMe₂ ); MS (EI) M/z no M⁺, 377 (6, M⁺-CH₃), 335 (26,M⁺-t-Bu), 253 (59), 209 (24), 173 (100), 161 (25), 133 (22); exact masscalculated for C₂₄H₄₄O₂SiNa (MNa⁺) 415.3008, found 415.3018.

Preparation of(20S,22E)-2-Methylene-19-nor-22-ene-1α,25-dihydroxyvitamin D₃ (13)

To a solution of phosphine oxide 11 (67 mg, 115 μmol) in anhydrous THF(500 μL) at −20° C. was slowly added PhLi (1.83 M in di-n-butylether, 75μL, 137 μmol) under argon with stirring. The solution turned deeporange. After 30 min the mixture was cooled to −78° C. and a precooled(−78° C.) solution of ketone 10 (26 mg, 66 μmol in anhydrous THF(300+300 μL) was slowly added. The mixture was stirred under argon at−78° C. for 3 h and at 0° C. for 18 h. Ethyl acetate was added, and theorganic phase was washed with brine, dried (Na₂SO₄) and evaporated. Theresidue was dissolved in hexane and applied on a Waters silica Sep-Pakcartridge (2 g). The cartridge was washed with hexane and hexane/ethylacetate (99.5:0.5) to give 19-norvitamin derivative 12 (39.54 mg, 79%yield).

UV (in hexane) λ_(max) 262.5, 253.0, 245.0 nm; ¹H NMR (400 MHz, CDCl₃) δ6.21 and 5.83 (each 1H, each d, J=11.1 Hz, 6- and 7-H), 5.38 (1H, ddd,J=15.4, 8.4, 6.8 Hz, 23-H), 5.29 (1H, dd, J=15.4, 8.7 Hz, 22-H), 4.97and 4.92 (each 1H, each s, ═CH₂), 4.42 (2H, m, 1β- and 3α-H), 2.81 (1H,dm, J=13.1 Hz, 9β-H), 2.52 (1H, dd, J=13.2, 5.8 Hz, 10α-H), 2.46 (1H,dd, J=12.7, 4.3 Hz, 4α-H), 2.33 (1H, dd. J=13.2, 2.4 Hz, 10β-H), 2.17(1H, dd, J=12.7, 8.4 Hz, 4β-H), 1.16 (6H, s, 26,27-H₆), 0.93 (3H, d,J=6.4 Hz, 21-H₃), 0.895 (9H, s, Si-t-Bu), 0.865 (9H, s, Si-t-Bu), 0.855(9H, s, Si-t-Bu), 0.518 (3H, s, 18-H₃), 0.0177 (3H, s, SiMe), 0.066 (9H,s, 3× SiMe), 0.047 (3H, s, SiMe), 0.025 (3H, s, SiMe); ¹³C NMR (100 MHz)δ 152.99 (s, C-2), 141.32 (s, C-8), 139.40 (d, C-22), 132.63 (s, C-5),124.25 (d, C-23), 122.42 (d, C-6), 116.00 (d, C-7), 106.24 (t, ═CH₂),73.78 (s, C-25), 72.53 and 71.63 (each d, C-1 and C-3), 56.67 (d), 56.20(d), 48.29 (t), 47.61 (t), 45.76 (s, C-13), 40.91 (d), 39.86 (t), 38.55(t), 29.83 and 29.62 (each q, C-26 and C-27), 28.77 (t), 27.41 (t),25.84 (q, 2× SiCMe₃ ), 25.78 (q, SiCMe₃ ), 23.23 (t), 22.11 (t), 21.57(q, C-21), 18.25 (s, SiCMe₃), 18.16 (s, SiCMe₃), 18.07 (s, SiCMe₃),12.11 (q, C-18), −2.04 (q, 2× SiMe), −4.86 (q, 2× SiMe), −4.90 (q,SiMe), −5.10 (q, SiMe); exact mass calculated for C₄₅H₈₄O₃Si₃Na (MNa⁺)779.5626, found 779.5651.

The protected vitamin 12 (39.44 mg, 52 μmol) was dissolved in THF (5 mL)and acetonitrile (3 mL). A solution of aq. 48% HF in acetonitrile (1:9ratio, 5 mL) was added at 0° C. and the resulting mixture was stirred atroom temperature for 11 h. Saturated aq. NaHCO₃ solution was added andthe reaction mixture was extracted with ethyl acetate. The combinedorganic phases were washed with brine, dried (Na₂SO₄) and concentratedunder reduced pressure. The residue was diluted with 2 mL ofhexane/ethyl acetate (8:2) and applied on a Waters silica Sep-Pakcartridge (2 g). An elution with hexane/ethyl acetate (75:25) gave thecrude product 13 (18 mg). The vitamin 13 was further purified bystraight phase HPLC [9.4×250 mm Zorbax Sil column, 5 mL/min,2-propanol/hexane (15:85) solvent system, R_(t)=6.46 min.] and then byreverse phase HPLC [9.4×250 mm Zorbax RX-C18 column, 3 mL/min,methanol/water (85:15) solvent system, R_(t)=10.19 min] to give acolorless oil (12.745 mg, 59% yield):

UV (in EtOH)λ_(max) 261.0, 252.0, 244.5 nm; ¹H NMR (400 MHz, CDCl₃)δ6.35 and 5.88 (1H and 1H, each d, J=11.2 Hz, 6- and 7-H), 5.44 (2H, m,22-H and 23-H), 5.11 and 5.09 (each 1H, each s, ═CH₂), 4.48 (2H, m, 1β-and 3α-H), 2.84 (1H, dd, J=13.3, 4.4 Hz, 10β-H), 2.80 (1H, br d, J=14.2Hz, 9β-H), 2.56 (1H, dd, J=13.4, 3.6 Hz, 4α-H), 2.32 (1H, dd, J=13.4,6.0 Hz, 4β-H), 2.28 (1H, dd, J=13.3, 8.4 Hz, 10α-H), 1.20 (6H, d, J=1.2Hz, 26,27-H₆), 0.95 (3H, d, J=6.6 Hz, 21-H₃), 0.528 (3H, s, 18-H₃); ¹³CNMR (100 MHz) δ151.96 (s, C-2), 143.27 (s, C-8), 141.71 (d, C-22),130.45 (s, C-5), 124.14 (d, C-6), 122.64 (d, C-23), 115.26 (d, C-7),107.69 (t, ═CH₂), 71.76 (d, C-1), 70.75 (s, C-25), 70.60 (d, C-3), 56.54(d), 56.13 (d), 46.90 (t), 45.80 (s, C-13), 45.74 (t), 40.72 (d), 39.80(t), 38.11 (t), 29.11 and 29.05 (each q, C-26 and C-27), 28.91 (t),27.26 (t), 23.24 (t), 22.09 (t), 21.61 (q, C-21), 12.28 (q, C-18); MS(EI) m/z 414 (37, M⁺), 396 (4, M⁺-H₂O), 381 (1, M⁺-H₂O—CH₃), 378 (1,M⁺-2H₂O), 356 (2), 311 (4), 287 (12), 269 (21), 251 (26), 194 (42), 147(41), 135 (100); exact mass calculated for C₂₇H₄₂O₃(M⁺) 414.3134, found414.3142.

Biological Activity of(20S,22E)-2-methylene-19-nor-22-ene-1α,25-dihydroxyvitamin D₃ Analogs

The introduction of a methylene group to the 2-position, as well as ahydroxyl substituent attached to the 25-position (C-25) in the sidechain, and having the methyl group normally located at the 21 position(C-21) in the side chain in its epi or S-configuration, and theintroduction of a double bond located between carbon atoms 22 and 23(C-22 and C-23) in the side chain, had little effect on binding of N-23to the full length recombinant rat vitamin D receptor, as compared to1α,25-dihydroxyvitamin D₃. The compound N-23 bound with the sameaffinity to the nuclear vitamin D receptor as compared to the standard1,25-(OH)₂D₃ (FIG. 1). It might be expected from these results thatcompound N-23 would have equivalent biological activity. Surprisingly,however, compound N-23 is a highly selective analog with uniquebiological activity.

FIG. 5 shows that N-23 has relatively low ability to increase intestinalcalcium transport activity in vivo at low dosages. It clearly has lowerpotency in vivo as compared to that of 1,25-dihydroxyvitaminD₃(1,25(OH)₂D₃), the natural hormone, in stimulating intestinal calciumtransport, especially at the recommended lower doses.

FIG. 4 demonstrates that N-23 has significant bone calcium mobilizationactivity, as compared to 1,25(OH)₂D₃. N-23 demonstrated slightly morebone calcium mobilization activity than 1,25(OH)₂D₃ (6.8 mg/dL of N-23versus 6.1 mg/dL of 1,25(OH)₂D₃ at 260 pmol dosage). Thus, N-23 clearlyis somewhat more effective in mobilizing calcium from bone as comparedto 1,25(OH)₂D₃.

FIGS. 4 and 5 thus illustrate that N-23 may be characterized as beingless potent than 1,25(OH)₂D₃ in promoting intestinal calcium transportactivity, but being slightly more potent than 1,25(OH)₂D₃ in promotingbone calcium mobilization activity.

FIG. 2 illustrates that N-23 is about 10 times more potent than1,25(OH)₂D₃ on HL-60 cell differentiation, i.e. causing thedifferentiation of HL-60 cells into monocytes. Thus, N-23 may be anexcellent candidate for the treatment of a cancer, especially againstleukemia, colon cancer, breast cancer, skin cancer and prostate cancer.

FIG. 3 illustrates that in bone cells the compound N-23 is about 10times more potent than 1,25(OH)₂D₃ in increasing transcription of the24-hydroxylase gene. This result, together with the cell differentiationactivity of FIG. 2, suggests that N-23 will be very effective intreating the above referred to cancers because it has direct cellularactivity in causing cell differentiation, gene transcription, and insuppressing cell growth.

Experimental Methods

Vitamin D Receptor Binding

Test Material

Protein Source

Full-length recombinant rat receptor was expressed in E. coli BL21 (DE3)Codon Plus RIL cells and purified to homogeneity using two differentcolumn chromatography systems. The first system was a nickel affinityresin that utilizes the C-terminal histidine tag on this protein. Theprotein that was eluted from this resin was further purified using ionexchange chromatography (S-Sepharose Fast Flow). Aliquots of thepurified protein were quick frozen in liquid nitrogen and stored at −80°C. until use. For use in binding assays, the protein was diluted inTEDK₅₀ (50 mM Tris, 1.5 mM EDTA, pH7.4, 5 mM DTT, 150 mM KCl) with 0.1%Chaps detergent. The receptor protein and ligand concentration wereoptimized such that no more than 20% of the added radiolabeled ligandwas bound to the receptor.

Study Drugs

Unlabeled ligands were dissolved in ethanol and the concentrationsdetermined using UV spectrophotometry (1,25(OH)₂D₃: molar extinctioncoefficient=18,200 and λ_(max)=265 nm; Analogs: molar extinctioncoefficient=42,000 and λ_(max)=252 nm). Radiolabeled ligand(³H-1,25(OH)₂D₃, ˜159 Ci/mmole) was added in ethanol at a finalconcentration of 1 nM.

Assay Conditions

Radiolabeled and unlabeled ligands were added to 100 mcl of the dilutedprotein at a final ethanol concentration of ≦10%, mixed and incubatedovernight on ice to reach binding equilibrium. The following day, 100mcl of hydroxylapatite slurry (50%) was added to each tube and mixed at10-minute intervals for 30 minutes. The hydroxylapaptite was collectedby centrifugation and then washed three times with Tris-EDTA buffer (50mM Tris, 1.5 mM EDTA, pH 7.4) containing 0.5% Titron X-100, After thefinal wash, the pellets were transferred to scintillation vialscontaining 4 ml of Biosafe II scintillation cocktail, mixed and placedin a scintillation counter. Total binding was determined from the tubescontaining only radiolabeled ligand.

HL-60 Differentiation

Test Material

Study Drugs

The study drugs were dissolved in ethanol and the concentrationsdetermined using UV spectrophotometry. Serial dilutions were prepared sothat a range of drug concentrations could be tested without changing thefinal concentration of ethanol (≦0.2%) present in the cell cultures.

Cells

Human promyelocytic leukemia (HL60) cells were grown in RPMI-1640 mediumcontaining 10% fetal bovine serum. The cells were incubated at 37° C. inthe presence of 5% CO₂.

Assay Conditions

HL60 cells were plated at 1.2×10⁵ cells/ml. Eighteen hours afterplating, cells in duplicate were treated with drug. Four days later, thecells were harvested and a nitro blue tetrazolium reduction assay wasperformed (Collins et al., 1979; J. Exp. Med. 149:969-974). Thepercentage of differentiated cells was determined by counting a total of200 cells and recording the number that contained intracellularblack-blue formazan deposits. Verification of differentiation tomonocytic cells was determined by measuring phagocytic activity (datanot shown).

In Vitro Transcription Assay

Transcription activity was measured in ROS 17/2.8 (bone) cells that werestably transfected with a 24-hydroxylase (24Ohase) gene promoterupstream of a luciferase reporter gene (Arbour et al., 1998). Cells weregiven a range of doses. Sixteen hours after dosing the cells wereharvested and luciferase activities were measured using a luminometer.

RLU=relative luciferase units.

Intestinal Calcium Transport and Bone Calcium Mobilization

Male, weanling Sprague-Dawley rats were placed on Diet 11 (0.47% Ca)diet +AEK oil for one week followed by Diet 11 (0.02% Ca)+AEK oil for 3weeks. The rats were then switched to a diet containing 0.47% Ca for oneweek followed by two weeks on a diet containing 0.02% Ca. Doseadministration began during the last week on 0.02% calcium diet. Fourconsecutive ip doses were given approximately 24 hours apart.Twenty-four hours after the last dose, blood was collected from thesevered neck and the concentration of serum calcium determined as ameasure of bone calcium mobilization. The first 10 cm of the intestinewas also collected for intestinal calcium transport analysis using theeverted gut sac method.

Interpretation of Data

Summary of Biological Findings. This compound N-23 binds the VDR withthe same affinity as the native hormone, and can be considered to beequally potent as 1,25(OH)₂D₃ in this activity. N-23 also displaysapproximately 10 times more cell differentiation activity and about 10times more in vitro gene transcription activity compared to 1,25(OH)₂D₃.While this compound is more potent compared to 1,25(OH)₂D₃ in vitro, itshows slightly more activity in vivo on bone calcium mobilizationcompared to the native hormone, and slightly less activity in vivo inpromoting intestinal calcium transport compared to the native hormone.N-23 is a potentially valuable compound for therapeutic development asit has higher potency in mobilizing calcium from bone storage and lowerpotency in stimulating active calcium transport in the gut, but higherpotency in cell differentiation potentially resulting in a compound witha bigger safety window than has previously been generated. Because thiscompound exhibits relatively significant cell differentiation andtranscriptional activity, but relatively low calcemic activity on bone,it might be useful for treating patients with various types of cancers,especially for the treatment of leukemia, colon cancer, breast cancer,skin cancer and prostate cancer. N-23 might not only be useful in thetreatment of the above listed cancers, but also in the prevention of theabove listed cancers.

VDR binding, HL60 cell differentiation, and transcription activity. N-23(K_(i)=6×10⁻¹¹M) has about the same activity as the natural hormone1α,25-dihydroxyvitamin D₃ (K_(i)=4×10⁻¹¹M) in its ability to competewith [³H]-1,25(OH)₂D₃ for binding to the full-length recombinant ratvitamin D receptor (FIG. 1), N-23 displays about 10 times more activity(EC₅₀=1×10⁻¹⁰M) in its ability (efficacy or potency) to promote HL-60cell differentiation as compared to 1α,25-dihydroxyvitamin D₃(EC₅₀=2×10⁻⁹M) (See FIG. 2). Also, compound N-23 (EC₅₀₋=2×10⁻¹¹M) hasabout 10 times more transcriptional activity in bone cells than1α,25-dihydroxyvitamin D₃ (EC₅₀=2×10⁻¹⁰M) (See FIG. 3). These resultssuggest that N-23 will have significant activity as an anti-cancer agentand will be very effective because it has direct cellular activity incausing cell differentiation, gene transcription, and in suppressingcell growth.

Calcium mobilization from bone and intestinal calcium absorption invitamin D-deficient animals. Using vitamin D-deficient rats on a lowcalcium diet (0.02%), the activities of N-23 and 1,25(OH)₂D₃ inintestine and bone were tested. As expected, the native hormone(1,25(OH)₂D₃ increased serum calcium levels at all dosages (FIG. 4). Thestudy repotted in FIG. 4 shows that N-23 has significant activity inmobilizing calcium from bone. The administration of 260 pmol/day of N-23for 4 consecutive days caused mobilization of bone calcium (6.8 mg/dL)and the native hormone 1,25(OH)₂D₃ had significant activity at 260pmol/day where a substantial effect was seen (6.1 mg/dL).

Intestinal calcium transport was evaluated in the same group of animalsusing the everted gut sac method (FIG. 5). The study reported in FIG. 5shows N-23 has less intestinal calcium transport activity than1,25(OH)₂D₃. Administration of 32 pmol/day of N-23 for 4 consecutivedays resulted in less activity as compared to 1,25(OH)₂D₃ at the same 32pmol/day dosage.

These results show that the compound N-23 promotes intestinal calciumtransport in a dose dependent manner. Thus, it may be concluded thatN-23 has lower intestinal calcium transport activity to that of1,25(OH)₂D₃ at the recommended lower doses.

These results further illustrate that N-23 is an excellent candidate fornumerous human therapies as described herein, N-23 is an excellentcandidate for treating a cancer because: (1) it has significant VDRbinding, transcription activity and cellular differentiation activity;(2) it has lower risk of hypercalcemic unlike 1,25(OH)₂D₃; and (3) it iseasily synthesized.

For prevention and/or treatment purposes, the compounds of thisinvention defined by formula I, particularly N-23, may be formulated forpharmaceutical applications as a solution in innocuous solvents, or asan emulsion, suspension or dispersion in suitable solvents or carriers,or as pills, tablets or capsules, together with solid carriers,according to conventional methods known in the art. Any suchformulations may also contain other pharmaceutically-acceptable andnon-toxic excipients such as stabilizers, anti-oxidants, binders,coloring agents or emulsifying or taste-modifying agents.

The compounds of formula I and particularly N-23, may be administeredorally, topically, parenterally, rectally, nasally, sublingually ortransdermally. The compound is advantageously administered by injectionor by intravenous infusion or suitable sterile solutions, or in the formof liquid or solid doses via the alimentary canal, or in the form ofcreams, ointments, patches, or similar vehicles suitable for transdermalapplications. A dose of from 0.01 μg to 1000 μg per day of the compoundsI, particularly N-23, preferably from about 0.1 μg to about 500 μg perday, is appropriate for prevention and/or treatment purposes, such dosebeing adjusted according to the disease to be treated, its severity andthe response of the subject as is well understood in the art. Since thecompound exhibits specificity of action, each may be suitablyadministered alone, or together with graded doses of another activevitamin D compound—e.g. 1α-hydroxyvitamin D₂ or D₃, or1α,25-dihydroxyvitamin D₃—in situations where different degrees of bonemineral mobilization and calcium transport stimulation is found to beadvantageous.

Compositions for use in the above-mentioned treatments comprise aneffective amount of the compounds I, particularly N-23, as defined bythe above formula I and Ia as the active ingredient, and a suitablecarrier. An effective amount of such compound for use in accordance withthis invention is from about 0.01 μg to about 1000 μg per gm ofcomposition, preferably from about 0.1 μg to about 500 μg per gram ofcomposition, and may be administered topically, transdermally, orally,rectally, nasally, sublingually, or parenterally in dosages of fromabout 0.01 μg/day to about 1000 μg/day, and preferably from about 0.1μg/day to about 500 μg/day.

The compounds I, particularly N-23, may be formulated as creams,lotions, ointments, topical patches, pills, capsules or tablets,suppositories, aerosols, or in liquid form as solutions, emulsions,dispersions, or suspensions in pharmaceutically innocuous and acceptablesolvent or oils, and such preparations may contain in addition otherpharmaceutically innocuous or beneficial components, such asstabilizers, antioxidants, emulsifiers, coloring agents, binders ortaste-modifying agents.

The compounds I, particularly N-23, may be advantageously administeredin amounts sufficient to effect the differentiation of promyelocytes tonormal macrophages Dosages as described above are suitable, it beingunderstood that the amounts given are to be adjusted in accordance withthe severity of the disease, and the condition and response of thesubject as is well understood in the art.

The formulations of the present invention comprise an active ingredientin association with a pharmaceutically acceptable carrier therefore andoptionally other therapeutic ingredients. The carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulations and not deleterious to the recipient thereof.

Formulations of the present invention suitable for oral administrationmay be in the form of discrete units as capsules, sachets, tablets orlozenges, each containing a predetermined amount of the activeingredient; in the form of a powder or granules; in the form of asolution or a suspension in an aqueous liquid or non-aqueous liquid; orin the form of an oil-in-water emulsion or a water-in-oil emulsion.

Formulations for rectal administration may be in the form of asuppository incorporating the active ingredient and carrier such ascocoa butter, or in the form of an enema.

Formulations suitable for parenteral administration convenientlycomprise a sterile oily or aqueous preparation of the active ingredientwhich is preferably isotonic with the blood of the recipient.

Formulations suitable for topical administration include liquid orsemi-liquid preparations such as liniments, lotions, applicants,oil-in-water or water-in-oil emulsions such as creams, ointments orpastes; or solutions or suspensions such as drops; or as sprays.

For nasal administration, inhalation of powder, self-propelling or sprayformulations, dispensed with a spray can, a nebulizer or an atomizer canbe used. The formulations, when dispensed, preferably have a particlesize in the range of 10 to 100μ.

The formulations may conveniently be presented in dosage unit form andmay be prepared by any of the methods well known in the art of pharmacy.By the term “dosage unit” is meant a unitary, i.e. a single dose whichis capable of being administered to a patient as a physically andchemically stable unit dose comprising either the active ingredient assuch or a mixture of it with solid or liquid pharmaceutical diluents orcarriers.

We claim:
 1. A compound having the formula::

where X₁, X₂ and X₃, which may be the same or different, are eachselected from hydrogen or a hydroxy-protecting group.
 2. The compound ofclaim 1 wherein X₃ is hydrogen.
 3. The compound of claim 1 wherein X₁ ishydrogen.
 4. The compound of claim 1 wherein X₁, X₂ and X₃ are allt-butyldimethylsilyl.
 5. A pharmaceutical composition containing aneffective amount of at least one compound as claimed in claim 1 togetherwith a pharmaceutically acceptable excipient.
 6. The pharmaceuticalcomposition of claim 5 wherein said effective amount comprises fromabout 0.01 μg to about 1000 μg per gram of composition.
 7. Thepharmaceutical composition of claim 5 wherein said effective amountcomprises from about 0.1 μg to about 500 μg per gram of composition. 8.A compound having the formula:

and named (20S,22E)-2-Methylene-19-nor-22-ene-1α,25-dihvdroxyvitamin D₃.9. A pharmaceutical composition containing an effective amount of(20S,22E)-2-Methylene-19-nor-22-ene-1α,25-dihydroxyvitamin D₃ togetherwith a pharmaceutically acceptable excipient.
 10. The pharmaceuticalcomposition of claim 9 wherein said effective amount comprises fromabout 0.01 μg to about 1000 μg per gram of composition.
 11. Thepharmaceutical composition of claim 9 wherein said effective amountcomprises from about 0.1 μg to about 500 μg per gram of composition, 12.A method of treating a disease selected from the group consisting ofleukemia, colon cancer, breast cancer, skin cancer or prostate cancercomprising administering to a subject with said disease an effectiveamount of a compound having the formula:

where X₁, X₂ and X₃ which may be the same or different, are eachselected from hydrogen or a hydroxyl-protecting group.
 13. The method ofclaim 12 wherein the compound is administered orally,
 14. The method ofclaim 12 wherein the compound is administered parenterally.
 15. Themethod of claim 12 wherein the compound is administered transdermally.16. The method of claim 12 wherein the compound is administeredrectally.
 17. The method of claim 12 wherein the compound isadministered nasally,
 18. The method of claim 12 wherein the compound isadministered sublingually.
 19. The method of claim 12 wherein thecompound is administered in a dosage of from about 0.01 μg/day to about1000 μg/day.
 20. The method of claim 12 wherein the compound is(20S,22E)-2Methylene-19-nor-22-ene-1α,25-dihydroxyvitamin D₃ having theformula: